Evaluation of antigen-specific T-cell responses with a miniaturized and automated method.
The evaluation of antigen-specific T-cell responses is helpful for both research and clinical settings. Several techniques can enumerate antigen-responsive T cells or measure their products, but they require remarkable amounts of peripheral blood mononuclear cells (PBMCs). Since screening numerous antigens or testing samples from pediatric or lymphopenic patients is hampered in clinical practice, we refined a miniaturized, high-throughput assay for T-cell immunity. Antigens and cells in 10-microl volumes were dispensed into 1,536-well culture plates precoated with anti-gamma interferon (anti-IFN-gamma) antibodies. After being cultured, the wells were developed by enzyme-linked immunosorbent assay for bound cytokine. Miniaturization and automation allowed quantitation of antigen-specific responses on 10(4) PBMCs. This method was applied for epitope mapping of mycobacterial antigens and was used in the clinic to evaluate T-cell immunity to relevant opportunistic pathogens by using small blood samples. A comparison with conventional methods showed similar sensitivity. Therefore, current flow cytometric methods that provide information on frequency and phenotype of specific T cells can be complemented by this assay that provides extensive information on cytokine concentrations and profiles and requires 20- to 50-fold fewer PBMCs than other analytical methods.
Li Pira
Giuseppina
G
Laboratory of Cellular Immunology, Advanced Biotechnology Center, Largo Benzi 10, 16132 Genoa, Italy. lipira@email.it
Ivaldi
Federico
F
Dentone
Chiara
C
Righi
Elda
E
Del Bono
Valerio
V
Viscoli
Claudio
C
Koopman
Gerrit
G
Manca
Fabrizio
F
D000906:Antibodies / Q000276:immunology; D000941:Antigens / Q000276:immunology*; D001331:Automation; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016207:Cytokines / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation; D018604:Epitope Mapping / Q000295:instrumentation / Q000379:methods*; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology; D008904:Miniaturization; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000276:immunology*
D000906:Antibodies; D000941:Antigens; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte; D007371:Interferon-gamma